ABSTRACT Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer in the United States. Even with the recent approval of immune modulating therapies, the overall survival rate for patients that respond poorly to conventional therapy remains at 40-50%, depending on subsite. We anticipate that significant improvements to patient survival will require the development of precision medicine protocols, in which treatments are designed to target the specific disease process of the individual patient. Immune checkpoint inhibition represents one potential targeted strategy, and pembrolizumab, which inhibits the interaction of the Programmed Death-1 (PD-1) receptor with its ligands, was approved for treatment of metastatic and recurrent HNSCC in 2016. However, an objective response rate of only 18% was reported in phase II trials of pembrolizumab, and few biomarkers reliably predict responses. We believe a more detailed understanding of the mechanisms underlying expression of the immunosuppressive Programmed Death Ligand 1 (PD-L1), will aid in patient selection for anti-PD-1 therapy and provide new avenues to improve response rates and duration. Our preliminary data suggest that the fibroblast growth factor receptor (FGFR) family may participate in PD-L1 regulation in specific subsets of HNSCC models, highlighting the potential for targetable oncogenic drivers to modulate this immunosuppressive pathway. Our central hypothesis is that genetic and immune biomarkers can predict immune checkpoint response in advanced or recurrent HNSCC, some of which (e.g. FGFR1) serve as molecular targets to alter the immunogenicity of tumor cells. Thus, this proposal has two aims that extend our initial observation of a relationship between the FGFR family and PD-L1 to define a mechanism and investigate combination FGFR and PD-1/PD-L1 inhibition as a potential therapeutic strategy. To expand this concept further, we have generated HNSCC cell lines infected with the Genome CRISPR Knock-Out (GeCKO) library from which we will first aim to select genetic knockouts conferring alterations in PD-L1 presentation on the cell surface, thereby implicating pathways involved in PD- L1 regulation. Using in vitro and in vivo models, we then aim to assess the mechanistic role of these pathways in modulating PD-L1 to identify novel therapeutic targets that may contribute to immunosuppression. By defining pathways regulating the PD-1/PD-L1 immune checkpoint, this proposal has the immediate potential to help improve upon current immunotherapy protocols for HNSCC to improve overall patient survival.